Fluorescence technique for on-line monitoring of state of hydrogen-producing microorganisms

ABSTRACT

In situ fluorescence method to monitor state of sulfur-deprived algal culture&#39;s ability to produce H 2  under sulfur depletion, comprising: a) providing sulfur-deprived algal culture; b) illuminating culture; c) measuring onset of H 2  percentage in produced gas phase at multiple times to ascertain point immediately after anerobiosis to obtain H 2  data as function of time; and d) determining any abrupt change in three in situ fluorescence parameters; i) increase in F t  (steady-state level of chlorophyll fluorescence in light adapted cells); ii) decrease in F m′ , (maximal saturating light induced fluorescence level in light adapted cells); and iii) decrease in ΔF/F m ′=(F m ′−F t )/F m ′ (calculated photochemical activity of photosystem II (PSII) signaling full reduction of plastoquinone pool between PSII and PSI, which indicates start of anaerobic conditions that induces synthesis of hydrogenase enzyme for subsequent H 2  production that signal oxidation of plastoquinone pool asmain factor to regulate H 2  under sulfur depletion.

CONTRACTUAL ORIGIN OF THE INVENTION

The United States Government has rights in this invention pursuant toContract No. DE-AC36-99GO10337 between the United States Department ofEnergy and the Midwest Research Institute.

This application is filed under Rule 371 as a National Stage from PCT/US02/12576 filed Apr. 19, 2002.

BACKROUND OF THE INVENTION

1. Field of the Invention

The invention relates to providing a non-destructive, external orremote, on-line way to monitor the physiological state of stressed(inclusive of nutrient stress and sulfur-deprivation stress) oxygenicphotosynthetic microorganisms that have a hydrogenase in situ withregard to their ability to produce H₂ gas without having to rely onelectrodes or other sensors placed in direct contact with the culturemedium.

2. Description of the Prior Art

The ability of eukaryotic microalgae to produce H₂ after a dark,anaerobic induction period was discovered about 60 years ago; however,the enzyme catalyzing this activity, a reversible Fe-hydrogenase, isinactivated by trace amounts of O₂ that is co-evolved duringphotosynthesis. Thus, light-dependent H₂ production by green algae,including Chlamydomonas reinhardtii, is observed for only a very shortperiod of time after anaerobic induction. Several years ago, Wykoff etal. ¹ found that incubating cells deprived of inorganic sulfur for up to120 h, resulted in the progressive reduction of photosynthetic capacitydue to the inactivation of photosystem II (PSII) O₂-evolution activityby up to 95%. 1. 1998. Plant Physiol. V. 117. P. 129-139.

Subsequent work [Melis et al. ² and Ghirardi et al. ³ 4investigated thiseffect more thoroughly and showed that after 1-2 days of adaptation tosulfur-deprived conditions, C. reinhardtii cells could producevolumetric amounts of H₂ gas for a few days under continuousillumination. The key observation made was that, when the rate ofphotosynthetic O₂ evolution declined below the rate of cell respiration,the culture could make itself anaerobic. Under these conditions, thehydrogenase enzyme was activated and/or induced in the light, and theonset of H₂ production followed after several hours. It has beendemonstrated that the process involved the sequential transition throughthe following five physiological phases: O₂-evolution, O₂-consumption,anaerobic adaptation, H₂-production, and termination phases. After thisoccurred, the culture could be regenerated by adding sulfate, prior toanother round of H₂ production. Nevertheless, the actual mechanismsinvolved in and the time-course for the decrease in PSII activity insulfur-deprived cells during cell transition to the anaerobic state aswell as the role of PSII in H₂ are not well established. 2. 2000. PlantPhysiol V. 122. P. 127-136.3 2000 a Trends Biotechnol. V. 18. P.506-5114

U.S. Pat. No. 5,372,784 discloses measurement of bacterial CO₂production in an isolated fluorophore by monitoring anabsorbance-regulated change of fluorescence. The fluorophore ispositioned to intersect the transmission light path, and indirectlymonitors absorbance or changes in the absorbance of a chromophoreencapsulated or isolated by a gas permeable polymetric matrix.

A biological treatment plant controlled by fluorescence sensors isdisclosed in U.S. Pat. No. 5,700,370. The method of using thesefluorescence sensors comprises:

monitoring the microbiological activity of the biological system and/orfluctuations thereof by on-line measurement of fluorescent emissionand/or variations therein for at least one characteristic biogenicfluorophore present in the mixed culture of microorganisms in the systemwhen irradiated with light and controlling one or several parameters ofthe process by using results from the measurement as measuredvariable(s) in an on-line automatization system.

U.S. Pat. No. 5,981,958 discloses a method and apparatus for detectingpathological and physiological change in plants. This invention providesan imaging fluorometer comprising a source of electromagnetic radiation,optical components to direct the radiation, excitation and emissionfilters, and an imaging-device. Radiation from the radiation source isused to excite fluorescence from a dark-adapted sample containingphotosynthetic components. This fluorescence is collected by the imagingdevice as a function of time and position within the sample. Excitationand emission filters limit the intensity and wavelengths of radiationincident on the sample and imaging device, respectively.

A method and material for determining relative abundance ofmicroorganisms in fixed populations is disclosed in U.S. Pat. No.6,107,033. The method entails:

-   -   1) providing a set of labeled in situ hybridization cluster        oligonucleotide probes;    -   2) hybridization of said probes with a sample of the mixed        population, and    -   3) quantitative analysis of the number of labeled        microorganisms.

German Patent, DE 19930865 discloses methods for detecting phytoplanktoncontent of natural water samples by chlorophyll fluorescencemeasurements. The measurements allow distinguishment among differentalgae groups on the basis of chlorophyll fluorescence measurements usinga measuring beam in combination with strong pulses of light withdifferent intensities, frequencies, phases, and wavelengths. The systemof this patent includes:

a first light source for producing a weak measuring light for excitingfluorescence in a selected portion of the sample volume;

a second light source for illuminating the remaining volume of thesample with a light intense enough to induce phototaxis (e.g., bydinoflagellates);

means for detecting and recording the time-dependent changes in light inthe sample volume and the portion of the sample undergoing measurement;and

means for comparing the changes to known values.

The time-dependent changes in fluorescence exhibit relative amplitudesand time constants, which are characteristic of certain dinoflagellatetypes.

Karukstis et al., in Photochemistry and Photobiology (1992) Vol. 55. No.1 pp. 125-132 disclose “ALTERNATIVE MEASURES OF PHOTOSYSTEM II ELECTRONTRANSFER INHIBITION IN ANTHRAQUINONE-TREATED CHLOROPLASTS” that compareelectron direct transport assays with an indirect fluorescence assay forbasic research studies.

An ON-LINE MONITORING OF CHLOROPHYLL FLUORESCENCEE TO ASSESS THE EXTENTOF PHOTOINHIBITION OF PHOTOSYNTHESIS INDUCED BY OXYGEN CONCENTRATION ANDLOW TEMPERATURE AND ITS EFFECT ON THE PRODUCTIVITY OF OUTDOOR CULTURESOF SPIRULINA PLATENSIS (CYANOBACTERIA) is disclosed by Torzilla et al.in J. Phycol. Vol. 34 (1998) pp. 504-510.

Polle et al. Biohydrogen II, (workshop) (2001) Meeting date 1999. pp.111-130 disclose MAXIMIZING PHOTOSYNTHETIC EFFICIENCIES AND HYDROGENPRODUCTION IN MICROALGA CULTURES by developing algal mutants withsmaller antenna sizes.

There is a need for providing a technique for on-line monitoring of thestate of hydrogen-producing microorganisms under aerobic and anaerobicconditions inside a closed photobioreactor system from an externalvantage point to gain information about the state of the culture withoutusing electrodes inserted directly into the culture medium, and therebypreclude the possibility of a source of (a) culture contamination andthe need to sterilize electrodes, and (b) gas, including oxygen andhydrogen, leaks, to produce a non-destructive, remote sensing procedure.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a process fordetermining the main factor(s) in regulating H₂ production under sulfurdepletion of oxygenic photosynthetic microorganisms that have ahydrogenase.

Another object of the present invention is to provide a process fordetermining the main factor(s) in regulating H₂ production under sulfurdepletion of oxygenic photosynthetic microorganisms that have ahydrogenase, by monitoring the culture from an external vantage point.

A further object of the present invention is to provide a process fordetermining the main factor(s) in regulating H₂ production under sulfurdepletion of oxygenic photosynthetic microorganisms that have ahydrogenase, by monitoring the culture from an external vantage point,without using electrodes or other type of sensor inserted directly intothe culture medium.

A yet further object of the present invention is to provide a processfor determining the main factor(s) in regulating H₂ production undersulfur depletion of oxygenic photosynthetic microorganisms that have ahydrogenase, by monitoring the culture free from a source of culturecontamination or gas leakage, that remotely senses the culture in amanner that is non-destructive to the sample.

In general, the invention process is accomplished by monitoring thephysiological state of sulfur-deprived algal cultures that progressthrough the physiological phases of: O₂ production, O₂ consumption,anaerobic adaptation, H₂ production, and termination, to ascertain anumber of significant and well-defined fluorescence parameter changesduring and between these phases; namely, to the effect that, at theexact time that sulfur-stressed algae become anaerobic, the followingthree in situ fluorescence parameters change dramatically and are usedto determine when and if a culture in a photobioreactor goes anaerobic:

-   -   F_(t) (the steady-state level of chlorophyll (Chl) fluorescence        in light-adapted cells) increases abruptly;    -   F_(m)′ (the maximal saturating light-pulse induced fluorescence        level in light-adapted cells) decreases abruptly; and    -   ΔF/F_(m)′=F_(m)′−F_(t))/F_(m)′ (the calculated photochemical        activity of photosystem II [PSII] under steady-state        illumination) decreases precipitously and abruptly.

The in situ fluorescence method for on-line monitoring of the state ofsulfur-deprived algal culture to ascertain the culture's ability toproduce H₂ under sulfur depletion entails:

-   -   a) providing a sample of sulfur-deprived algal culture        containing photosynthetic components;    -   b) illuminating the sample with artificial or natural        illumination;    -   c) determining the onset of H₂ photoproduction by measuring the        percentage of H₂ in a produced gas phase at multiple times to        ascertain the point immediately after the anerobiosis subsequent        to the physiological phases of O₂ production and O₂ consumption        sequence to obtain data regarding H₂ as a function of time; and    -   d) determining any abrupt change in the following three in situ        fluorescence parameters:        -   i) an abrupt increase in F_(t) (the steady-state level of            chlorophyll fluorescence in light adapted cells);        -   ii) an abrupt decrease in F_(m), (the maximal saturating            light induced fluorescence level in light adapted cells);            and        -   iii) a precipitous and abrupt decrease in            ΔF/F_(m)′=(F_(m)′−F_(t))/F_(m)′ (the calculated            photochemical activity of photosystem II (PSII)) that signal            the full reduction of the plastoquinone pool between PSII            and PSI, which indicates the start of anaerobic conditions            that in turn induces the synthesis of the hydrogenase enzyme            required for subsequent H₂ production, and thereafter            slowing down of the abrupt decrease and partial recovery of            Δ F/F_(m)′ signal at least partial oxidation of the            plastoquinone pool as the main factor to regulate H₂            production under sulfur depletion.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURES

FIG. 1 is a graph showing the effect of 10 μM DCMU on the rate of H₂photoproduction by cultures of sulfur-depleted C. reinhardtii cells,where the control cells are open circles and the DCMU-treated cells, areclosed articles. DCMU was added to treated cells at the arrow. Time zeroin this case represents the time after H₂ photoproduction starts.

FIG. 2 is a graph of a time course of physiological parameters and H₂production in C. reinhardtii cells during incubation undersulfur-deprived conditions. More particularly, FIG. 2A shows in situfluorescence parameters F_(t) (open squares), ΔF/F_(m)′ (solid circles),and ΔF_(m)′ (open triangles) and chlorophyll concentrations (Chl; solidtriangles) as a function of time, and FIG. 2B shows dissolved oxygen(pO₂, crosses), redox potential E_(h) (stars) and H₂ gas collected in aninverted graduate cylinder (solid squares). Incubation insulfur-deprived medium started at 0 h.

FIG. 3 is a graph showing in situ fluorescence parameters F_(t) (opensquares), ΔF/F_(m)′ (solid circles), and F_(m)′ (open triangles), aswell as pO₂ (crosses), E_(h) (stars) and H₂ content in the gas phase ofculture vessel (solid squares) in illuminated, sulfur-deprived, C.reinhardtii during the transition of the algae from aerobic to anaerobicconditions. Incubation in sulfur-deprived medium started at 0 h.

FIG. 4 is a graph showing changes in chlorophyll fluorescence parametersrecorded in a dark-adapted algal sample, which was removed anaerobicallyfrom a culture vessel 22 hours after the beginning of H₂ production.After 18 minutes of dark adaptation, the algal sample was aerated(arrow). F₀ (open squares), F_(m) (open triangles), and F_(v)/F_(m)(solid circles) were monitored periodically as a function of time afterremoval from the culture vessel.

FIG. 5 is a graph showing chlorophyll fluorescence induction curves incontrol algae at the start of sulfur deprivation (A) and in cellsremoved from a culture vessel anaerobically after 22 hours of H₂production (B, C, D). (B) Fluorescence kinetics were recorded while thecells were illuminated with saturating light after a 10-minute period ofdark adaptation. (C) Same as (B) except far red light (λ˜735 nm) wasturned on for one second prior to the measurement. (D) Same as (B)except that the sample was aerated just prior to measurement. The F₀level occurs at time zero.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT OF INVENTION

Chlamydomonas reinhardtii strain Dangeard C137+ was grown inTris-acetate-phosphate (TAP) medium (pH, 7.0) under continuouscool-white fluorescent light. Late-log cells were pelleted three timesby centrifiguation and re-suspended in the same medium, from whichinorganic sulfure (TAP-S) was omitted. The cells were incubated undercontinuous fluorescent illumation (200-250 μE m⁻² s⁻¹ PAR) incylindrical reactor vessels (600 ml) with a rotating glass rod andTeflon impellers for mixing. A magnetic bar was attached at the bottomof the rod to couple it to an external stirring motor, and the rate ofmixing was 60-80 rpm. The vessels were sealed using a silicon stopperwith an outlet tube inserted through it for gas collection. The gasproduced by the cells was collected in an inverted graduated glasscylinder by water displacement.

To determine the precise moment of H₂-photoproduction onset, we measuredthe percentage of H₂ contained in the gas phase (˜7 ml) on the top ofthe vessel immediately after anaerobiosis by gas chromatography. Thedissolved O₂ and redox potential of the medium were also measured.Chlorophyll concentrations were then obtained spectrophotometrically in95% ethanol cell extracts. The capacity for respiratory O₂ consumptionwas measured with a Clark electrode at 25° C. under aerobic conditions(17% or 170 μM O₂) after removing the samples from the culture vesseland equilibrating them with air for 5 minutes.

Chlorophyll fluorescence measurements were obtained with a portablePAM-2000 fluorometer using the weak modulated pulse-probe(Pulse-Amplitude-Modulation [PAM]) method. In situ measurements offluorescence yield (λ>710 nm) were done with an optical fiber probeaffixed closely onto the surface of the illuminated glass reactorvessel. The measurements were recorded every 15 minutes over the totalperiod of sulfur deprivation. A 0.8 s saturating actinic excitationpulse (λ<710 nm, 1200 μmol·m⁻²·s⁻² PAR) from an 8V/20 W halogen lamp wasapplied on top of weak modulated probe flashes (3 μs pulses from a 655nm light-emitting diode (LED) at frequencies of 600 Hz or 20 kHz.) forFIGS. 2-4, whereupon the following parameters were recorded: (a) F₀, thefluorescence yield of dark-adapted cells measured only in the presenceof weak probe flashes; (b) F_(m), the fluorescence yield of the samecells, following the application of the saturating actinic pulse; (c)F_(t), the fluorescence yield of the cells, exposed to the ambient,steady-state illumination from fluorescence lamps or natural lighting inthe reactor vessels, measured with the weak probe flashes; and (d)F_(m)′, the fluorescence yield of illuminated cells, as in (c),following application of the saturating actinic pulse. The efficiency ofphotochemical conversion of absorbed light energy (the photochemicalactivity) in PSII was calculated both after dark adaptation, whereF_(v)/F_(m)=(F_(m)−F₀)/F_(m), and under steady-state actinic lightillumination, where Δ F/F_(m)′=(F_(m)−F_(t))/F_(m)′. The kinetics offluorescence induction was also measured with the PAM-2000 fluorometer.Samples were removed anaerobically from the reactor vessel and placedinto a transparent plastic cuvette hermetically sealed with a stopper.Following a 10-minute dark, anaerobic adaptation period, the sampleswere illuminated with weak modulated probe flashes as above and actiniclight (655 nm, 250 μmol·m⁻² ·s⁻¹ PAR from a LED array for 2 s), and thekinetics of fluorescence induction were measured at λ>710 nm (FIG. 5).

As mentioned above the sulfur-deprivation process involvesO₂-production, O₂-consumption, anaerobic-adaptation, H₂-production andtermination phases. These phases proceed over different periods of time,depending on the residual sulfur concentration (at the time of sulfurremoval), chlorophyll and biomass content in the culture, pH of theculture medium, and other factors. This is why the beginning of theanaerobic phase is observed at different times after sulfur-deprivationin different experiments. The observed changes in cell parameters duringthe sulfur-deprivation process (excluding FIGS. 2 and 3) are expressedrelative to the indicated physiological stage of the cultures.

One of the most critical questions about the mechanism of H₂photoproduction in sulfur-deprived systems is the identification of thesource of the electrons for gas production. The electrons could comefrom storage products or from water (i.e., electrons from water aretransported from PSII to PSI), with subsequent reduction of ferredoxinand hydrogenase.

FIG. 1 shows the addition of DCMU (a potent inhibitor of electron flowat the level of Q_(B) [the secondary PSII quinone acceptor] on thereducing side of PSII) to a culture, after H₂ gas production hasstarted, abruptly decreases the rate of gas production by 80%. Thisdemonstrates that most of the electrons from H₂ production are comingfrom water and is consistent with the fact that large amounts of H₂ areonly produced in the light. The direct involvement of PSII-catalyzedwater oxidation activity by sulfur-deprived cells was furtherinvestigated using PAM fluorescence techniques.

During the O₂-production and consumption phases of sulfur deprivation(the first 15 hours in the experiment shown in FIG. 2A), the Chlconcentration was set at 11 μg/ml at the beginning of the experiment,and the in situ-monitored fluorescence parameter, F_(m)′, did not changesignificantly. However, since the fluorescence parameter, F_(t), slowlyincreased during this same period, the photochemical activity of PSII(ΔF/F_(m)′), declined from 0.57 in the middle of the O₂-production phaseto 0.44 at the end of the O₂-consumption phase (FIG. 2A). The observedchanges in F_(m)′, F_(t) and Δ F/F_(m)′ reflect the existence oflong-lived inactive states apparently generated in theQ_(B)-non-reducing centers of PSII. In fact, if samples of the algalsuspension were removed aerobically from the culture vessels at the endof the O₂-consumption phase and dark-adapted for different periods oftime, the fluorescence parameters, F₀ and F_(m), and the fluorescenceinduction curves did not change significantly during a 40-minuteincubation period (data not shown). This suggests that theQ_(B)-non-reducing state of PSII is long-lived in the dark once it isestablished.

The results are consistent qualitatively with previous observationsshowing that the capacity for O₂ production by PSII, the amount oflight-reducible Q_(A) ⁻, (reduced form of the primary PSII quinoneacceptor) and the rate of electron transport from PSII and PSI decreaseduring sulfur deprivation. However, it is noteworthy to point out thatthe experiments of Wykoff et al. and Melis et al. were done by removingsamples from a photobioreactor and assaying for O₂ evolution underaerobic conditions, whereas our results are from in situ measurements.

FIG. 2A also shows that, in this experiment at 15 and 16 h after thestart of sulfur deprivation, there was a rapid (<5 min) reduction of thein situ-measured photochemical yield of PSII (ΔF/F_(m)′) from 0.4 to0.1, which reflects a rapid down-regulation of PSII photochemicalactivity at this time. The loss of photochemical activity was due bothto a rapid reduction in F_(m)′ and a rapid increase in F_(t) It isimportant to note that in FIG. 3 (an expanded view of data from FIGS. 2Aand B) the observed rapid reduction of PSII photochemical activity(ΔF/F_(m)′) began at the exact time that the O₂ concentration in theculture suspension reached zero. The redox potential of the culturemedium also started to decline around this time (FIGS. 2B and 3) but notnearly so fast as the O₂ concentration and PSII photochemical activity.At about one hour of anerobiosis, the PSII photochemical activitydropped to a minimum value (0.05). After that point, it graduallyincreased over the next 2 hours and reached a maximum value of 0.085(FIGS. 2A and 3). No additional large changes were observed over 20 morehours of anerobiosis and H₂ production. If at this point (21 h after thebeginning of anaerobiosis), an algal sample was removed anaerobicallyfrom the reactor vessel and placed into an anaerobic cuvette for 18minutes in the dark, the PSII activity (F_(v)/F_(m)) of the dark-adaptedcells increased rapidly from 0.07 to 0.14 when the cell suspension wassubsequently aerated (FIG. 4). Following the aeration, the F_(v)/F_(m)ratio increased slowly up to a value of 0.22 after an additional hour ofaerobic incubation. This clearly demonstrates that the rapid in situanaerobic inactivation of PSII photochemical activity is partiallyreversible by O²⁻ in contrast to the slower aerobic inactivation of PSIIobserved during the O²⁻ consumption stage of sulfur deprivation.

Volumetric measurements of H₂ gas accumulation, such as those presentedin FIG. 2B, do not give the exact time of the start of H₂photoproduction. To determine the exact onset of H₂ production, wemeasured the concentration of H₂ in the gas phase of the reactor vessel(FIG. 3). Trace quantities of H₂ (0.002%) were detected as early as 10minutes after the onset anaerbiosis. This is reflected at the pointwhere the decrease in The ΔF/F_(m)′ slows dramatically. The rate of H₂accumulation in the gas phase increased from 0.04 μl of H₂ per minutewithin the first 10 minutes of anaerobiosis up to 1.0 μl per minute atthe end of the second hour of anaerobiosis. This was accomplished by amoderate increase in the ΔF/F_(m)′ value. Gas accumulation in theinverted graduate cylinder started after about 4 h of anaerobiosis (FIG.2B), and the rate of H₂ accumulation was constant until at least 22 hafter the beginning of anaerobiosis. These results suggest that thealgae actually start to produce H₂ very shortly after the establishmentof anaerobiosis. Apparently it takes a couple of hours to saturate thealgal suspension with H₂ (the solubility of H₂ in water is 17 ml per 1at 30° C.) and then build up enough gas pressure to displace the waterin the gas-collection system. We show earlier that, under differentinitial conditions of cell cultivation and/or less accurate measuringconditions, the in situ measured photochemical yield of PSII drops tozero after the rapid decrease in photochemical yield. In the currentstudy the observed recovery or up-regulation of PSII activity underanaerobic conditions correlates with the onset of H₂ production. Thiscorrelation confirms that PSII, and consequently residualwater-splitting function, is the main source of electrons of H₂production under these conditions.

As to the actual mechanisms responsible for the observed in situ changesin PSII activity in sulfur-deprived cultures, the initial slowinactivation of PSII capacity is believed to be due to the decreasedability of the cells to replace photodamaged D1 protein, thus leading tothe accumulation of Q_(B)-non-reducing PSII centers that cannot produceO₂. These changes result in the establishment of anaerobic conditions inthe reactor vessel in the light.

In addition to the slow inactivation of PSII capacity, we demonstratethat a new, rapid down-regulation of PSII photochemical activity occursjust as the culture becomes anaerobic (FIGS. 2 and 3). This rapiddown-regulation is not accompanied by a proportional loss of PSIIcapacity as measured by the capability of PSII to accumulate Q_(A)-or toevolve O₂ when the cells are exposed to aerobic conditions [FIG. 4]. Itis believed that the rapid in situ PSII down-regulation is a response ofthe algae to the over-reduction of the plastoquinone (PQ) pool, whichmust accompany the establishment of anerobiosis. Since there is no O₂ toreoxidize the PQ pool under anaerobic conditions and since PSI cannotfurther dispose of electrons from the pool (the hydrogenase is notactive yet and there is little rubisco to utilizephotosynthetically-generated electrons), PSII activity is reversiblydown-regulated. A similar mechanism, but slower rate has been used toexplain the irreversible loss of PSII activity in eukaryotic algaefollowing exposure to high temperatures or when deprived of nutrientsother than sulfur.

The evidence presented in FIG. 5 supports our suggestion that rapiddown-regulation of PSII is induced by over-reduction of the PQ poolunder anaerobic conditions. It is known that the rise from F₀ to theF_(i) level on the fluorescence induction curve corresponds to theportion of PSII centers that are unable to reduce Q_(B). A control,aerobic algal sample, removed from the aerobic culture vessel at thestart of sulfur deprivation (see FIG. 2), was examined first. In thissample (FIG. 5A), the F_(i) peak is followed by a slower rise offlorescence to the—maximum level, and this reflects the reduction of thePQ pool. In contrast, fluorescence-induction curves recordedanaerobically from anaerobic, dark-adapted cells, 22 h after the startof anaerobic phase (FIG. 5B), had a pronounced F_(i) peak but completelylacked a slow rise phase. The lack of the slow rise indicates that thePQ pool cannot be further reduced under these conditions. Accordingly,the F_(i) peak disappeared if the algal suspension was pre-illuminatedwith far red light (FIG. 5C) or aerated (FIG. 5D) prior to recording thefluorescence kinetics. In the former case, the PQ pool was oxidized byPSI, which preferentially absorbs far red light, and in the latter itwas oxidized by O₂ itself. The loss of the distinct F_(i) peak occursbecause oxidation of a small fraction of the PQ pool under theseconditions reoxidizes Q_(B) and this lowers the F_(i) level to that ofthe steady-state value on this time scale. Under anaerobic conditions,reduction of the PQ pool occurs by residual photosynthetic electrontransfer and/or by the transfer of reductants from anerobic substratedegradation in the chloroplast stroma to the PQ pool through theNAD(P)H-PQ oxidoreductase. In fact, the existence of such reductanttransfer in our samples is confirmed by incomplete (80%) suppression ofH₂ production after the addition of DCMU (FIG. 1).

The consumption of photosynethically produced O₂ during theH₂-production phase is essentially for maintaining the culture mediumanaerobic for the operation of the hydrogenase. Oxygen consumption in C.reinhardtii is catalyzed by three main oxidases: (a) the chloroplastPQ-oxidase; (b) the mitochondrial cytochrome c-oxidase; and (c) themitochondrial alternative oxidase. The existence of the respiratoryelectron transport process, chlororespiration, occurring in thechloroplasts of green algae explains the effect of respiratoryinhibitors on chlorophyll fluorescence transients. The chlororespiratorychain allows the transfer of reductants (NADPH), generated by substratedegradation in the chloroplast stroma, to the PQ pool via the NAD(P)H-PQoxidoreductase.

The PQ pool is further oxidized by dissolved O₂ through the action ofPQ-oxidase. In the mitochondria, NADH and FADH₂ generated by the KrebsCycle serve as the source of electrons for a membrane-bound electrontransport chain with O₂ as the final electron acceptor. The cytochromeoxidase transduces the production of 3 ATP molecules per pair ofelectrons transported. The alternative oxidase, on the other hand, isactually active under conditions that require less ATP/NADH since itsoperation generates only 2 ATP/pair of electrons. In both cases, O₂ isthe terminal electron acceptor. The mitochondrial cytochrome oxidase inC reinhardtii is inhibited by KCN, carbon monoxide, and sodium azide,while the alternative oxidase is specifically affected by salicylichydroxamic acid (SHAM). The putative chloroplast PQ oxidase, on theother hand, is not inhibited by SHAM and only slightly affected by KCN.By measuring the influence of two inhibitors, KCN and SHAM, on the ratesof dark respiration, we tried to estimate the relative contributions ofeach of the three oxidases at different times after sulfur deprivation.The actual contributions of the three oxidases to the scavenging O₂ insitu depend on many factors such as their relative K_(M)'s for O₂ andthe rate of diffusion of O₂ from the chloroplast to the mitochondria.Thus, the inhibitor studies that follow may not directly reflect theactual situation in the photobioreactor, but they do indicate thatchanges in the relative contribution of the three oxidases occur overthe course of sulfure deprivation.

Table 1 shows that sulfur-deprivation gradually inhibits the rates ofdark respiration in C. reinhardtii over a 100-h incubation period.

TABLE I Rate of % SHAM Putative % respiration % KCN inhibitionPQ-oxidase Phase of Sulfur (μmoles O₂/mg inhibition (% (% (% depletionChl⁻¹ × h⁻¹ CO) AO) CR) Start of sulfur 30 60 25 15 depletion Start of28 16 0 84 anaerobic phase Start of H₂ 22 36 13 51 production phaseDuring H₂ 18 8 38 54 production End of H₂ 6 17 100 0 production

Contributions to the mitochondrial cytochrome oxidase [CO],mitochondrial alternative oxidase [AO], and chlororespiration [CR] tothe total respiration rate of C. reinhardtii cells are also shown duringsulfur-deprivation. Respiration rates were measured with cells takenfrom the bioreactor at different times after sulfur depletion,equilibrated with air and incubated with the inhibitors for 5 min. Rateswere measured when the O₂ concentration in the medium was 17% O₂. Finalconcentrations of KCN and SHAM were 5 mM and 4 mM, respectively.

At the start of sulfur deprivation, the cytochrome oxidase and thealternative oxidase contributed, respectively, about 60 and 25% to theoverall respiration capacity of the cells measured in the presence of17% (170 μM) O₂. The remainder (about 15%) appears due to the activityof the chloroplast PQ-oxidase (Table I, last column),; however, itshould be noted that the specificity of the inhibitors is not 100%. Atthe start of the anaerobic phase, the activity of the two mitochrondialoxidases decreased substantially to 16 and 0% of the total respirationcapacity, respectively. This indicates a shift from mitochondrialrespiration to chlororespiration. The shift towards chlororespirationwas coupled to the shut-down of electron transport from PSII (FIGS. 2and 3) caused by the over-reduction of the PQ pool, and the completeremoval of O₂ from the sulfur-deprived culture medium. Once H₂ evolutionstarted and accompanying up-regulation of PSII began,photosynthetically-generated O₂ appeared to be consumed equally by themitochondrial (49%) and chloroplast (51%) oxidases (see Table I). Duringthe H₂-production phase, the contribution of the chlororespiratory PQoxidase remained unchanged. However, over the same period of time, thecontribution of the mitochondrial cytochrome oxidase declineddramatically while the alternative oxidase increased. At the end of theH₂-production phase, the mitochondrial respiratory activity was duemostly to the alternative oxidase. Thus, the inhibitor studies in TableI support the active role of chlororespiration in maintaining theanaerobic conditions in the chloroplast required for H₂ photoproduction,and they are also consistent with the idea that the alternative oxidaseis expressed preferentially under stress conditions. This differentialactivity has also been observed in phosphate- or nitrogen-depleted C.reinhardtii and tobacco cells.

From FIGS. 2 and 3, it can be seen that the sharp down-regulation ofPSII photochemical activity is reflected in an increase in F_(t), whichis the result of a sharp increase in the number of active PSII centersthat are unable to reduce Q_(B). Decreases in F_(m)′ are due toincreases in non-photochemical quenching of PSII, and one of the knownmechanisms responsible for non-photochemical quenching is the generationof a ΔpH gradient across the photosynthetic membrane. These fluorescencestudies show that the sharp drop in F_(m)′ at the time that O₂concentration in the culture reaches zero occurs at approximately thesame time that the PQ pool becomes over-reduced. Since the oxidized PQpool is the source of reductant for chlororespiration, the inhibitorstudies (Table I) appear to indicate that the chlororespiratory pathwaybegins to make a major contribution to O₂ removal from the culture atabout the same time. It therefore appears that the rapid decrease inPSII photochemical is also due to the enhancement of chlororespiration,accompanied by the generation of a gradient. The increase in ΔpH appearsto be the main functional mechanism of non-photochemical quenching underthe conditions responsible for the observed drop in F_(m)′.

The observed up-regulation of PSII activity that follows theestablishment of anaerobiosis in the culture vessel (FIGS. 2B and 3)appears to be the result of induction of reversible Fe-hydrogenaseenzyme activity, which catalyzes ferredoxin-dependent reduction ofprotons and releases H₂. In fact, the hydrogenase provides a sink forelectrons generated either by light-catalyzed water oxidation or bymetabolic oxidation of endogenous substrates, and its activity resultsin the partial oxidation of the intermediate photosynthetic carriers,including the PQ pool. Hence under anaerobic conditions, H₂ productionby sulfur-deprived cells allows the system to retain a fraction of thePSII reaction centers in a photochemically active state. The O₂ evolvedunder these conditions, as indicated, appear to be consumed in part bychlororespiration or by storage-product degradation linked tomitochondrial respiratory processes that utilize both the cytochromeoxidase and the alternative oxidase as the final electron acceptors.

In the context of the inventive process, it is important to note that,if only the redox potential is measured without measuring the dissolvedO₂ concentration, it is not possible to know exactly when the systemgoes anaerobic; however, the invention process measures the O₂concentration in the culture at the same time that the fluorescence ismeasured to ascertain the exact time the system goes anaerobic (requiredfor H₂ production). More specifically, the exact time the system goesanaerobic is when the fluorescence (F/F_(m)′) starts its abrupt drop,and this is the point where PSII activity is down-regulated.

In summarization, it is seen that changes of PSII activity in C.reinhardtii cells deprived of inorganic sulfur are characterized bycomplicated dynamics during the course of cell adaptation to thenutrient stress. We therefore conclude that (a) the redox state of thePQ pool is the primary factor in regulating the activity of anyremaining PSII water-splitting capacity by controlling the PSIIphotochemical activity in the algae during all phases of sulfurdeprivation and (b) most of the electrons used directly for H₂ gasproduction come from water. Thus, the redox state of the PQ pool dependson the relationship between the rates of photosynthesis,chlororespiration, respiration, and H₂ production.

1. An in situ fluorescence method for external on-line monitoring of thephysiological state a sulfur-deprived algal culture inside a closedphotobioreactor system to ascertain the culture's production of H₂ undersulfur depletion, comprising: a) providing a sample of sulfur-deprivedalgal culture containing photosynthetic components; b) illuminating saidsample with artificial or natural illumination; c) determining the onsetof H₂ photoproduction by measuring the percentage of H₂ in a producedgas phase at multiple times to ascertain the point immediately after theanerobiosis subsequent to the physiological phases of O₂ production andO₂ consumption sequence to obtain data regarding H₂ production as afunction of time; and d) determining any abrupt change in the followingthree in situ fluorescence parameters: i) an abrupt increase in F_(t)(the steady-state level of chlorophyll fluorescence in light adaptedcells); ii) an abrupt decrease in F_(m)′ (the maximal saturating lightinduced fluorescence level in light adapted cells); and' iii) aprecipitous and abrupt decrease in ΔF/F_(m)′=(F_(m)′−F_(t))/F_(m)′ (thecalculated photochemical activity of photosystem II (PSII)) that signalsthe full reduction of the plastoquinone pool between PSII and PSI, whichindicates the start of anaerobic conditions that in turn induces thesynthesis of the hydrogenase enzyme required for subsequent H₂production, and thereafter slowing down of the abrupt decrease andpartial recovery of ΔF/F_(m)′ signals at least partial oxidation of theplastoquinone pool as the main factor to regulate H₂ production undersulfur depletion.
 2. The method of claim 1 wherein said algal culture isany oxygenic photosynthetic microorganism that has a hydrogenase.
 3. Themethod of claim 2 wherein said oxygenic photosynthetic microorganismthat has a hydrogenase is green algae.
 4. The method of claim 3 whereinsaid green algae is selected from the group consisting of Chlamydomonasreinhardtii, Scenedesimus obligus and Chlorella vulgaris.
 5. The methodof claim 4 wherein said green algae is Chlamydomonas reinhardtii.
 6. Themethod of claim 5 wherein said abrupt increase in F_(t) is determinedusing a fluorometer employing a weak modulated pulse-probe fluorescencemethod.
 7. The method of claim 5 wherein said in situ measurement offluorescence is at or about λ>710 nm.
 8. The method of claim 7 whereinsaid in situ measurement of fluorescence is performed with an opticalfiber probe affixed onto a surface of an illuminated glass containingfluorescence excited sample or alternatively with a lens system.
 9. Themethod of claim 7 wherein said in situ measurement of fluorescence isperformed with a fluorometer set close to the edge of a bioreactor. 10.The method of claim 7 wherein said in situ measurement of fluorescenceis performed with a lens set close to the edge of the bioreactor. 11.The method of claim 8 wherein a saturated actinic excitation pulse isapplied on top of a weak modulated probe pulse.
 12. The method of claim11 wherein said saturated actinic excitation pulse is a 0.8 s pulse(about λ<710 nm, 1200 μmol·m⁻²·s⁻² PAR) from an 8 V/20W halogen lamp.13. The method of claim 11 wherein actinic light is about 655 nm, 250μmol·m⁻²·s⁻¹ PAR from a LED array for about 2 s for fluorescenceinduction.
 14. The method of claim 12 wherein said saturating actinicexcitation pulse is applied on top of a weak modulated probe thatflashes at about 3 μs pulses from a 655 nm light-emitting diode atfrequencies of from about 600 Hz or 20 kHz.
 15. The method of claim 14wherein efficiency of photochemical conversion of absorbed light energyin PSII is calculated after dark adaptation, whereF_(v)/F_(m)=(F_(m)−F_(o))/F_(m).
 16. The method of claim 14 whereinefficiency of photochemical conversion of absorbed light energy in PSIIis calculated under steady-state actinic light illumination, whereΔF/F_(m)′=(F_(m)′−F_(t))/F_(m)′.